Purine Salvage Pathway of Cryptosporidium Parvum

小隐孢子虫的嘌呤回收途径

• 批准号: 7569515
• 负责人: BUDDY ULLMAN
• 金额: $ 38.16万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-02-15 至 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7569515
• 关键词: AMP Deaminase; Acquired Immunodeficiency Syndrome; Address; Adenosine Kinase; Applications Grants; Biochemical; Biochemistry; Biological; Biological Assay; Categories; Cell Culture Techniques; Cells; Cellular biology; Chemicals; Cryptosporidiosis; Cryptosporidium parvum; Development; Disease; Dissection; Drug Delivery Systems; Eligibility Determination; Enzymes; Escherichia coli; Fluorescence; GMP synthase; Genes; Genetic; Genome; Genomics; Goals; Human; Infection; Intention; Investigation; Kinetics; Knock-out; Lesion; Ligase; Molecular; Molecular Biology; Molecular Models; Mutate; National Institute of Allergy and Infectious Disease; Nucleosides; Nutritional; Opportunistic Infections; Organism; Parasites; Parasitic Diseases; Pathway interactions; Pharmaceutical Preparations; Pharmacology; Property; Proteins; Purine Nucleotides; Purines; Reaction; Reagent; Recombinant Proteins; Recombinants; Research; Research Project Grants; Resolution; Role; Saccharomyces cerevisiae; Scheme; Structure; Structure-Activity Relationship; Substrate Specificity; Technology; Testing; Therapeutic; Toxoplasma gondii; Transgenic Organisms; Vaccines; Validation; base; biodefense; chemotherapy; drug discovery; enzyme structure; gene function; genetic selection; guanylate; high throughput screening; in vivo; inhibitor/antagonist; kinase inhibitor; molecular modeling; mutant; novel; nucleobase analog; nucleoside analog; overexpression; pathogen; purine; purine analog; repository; structural biology; therapeutic target; three dimensional structure; tool; waterborne; weapons

DESCRIPTION (provided by applicant): Amalgamating the tools of molecular biology, biochemistry, cell biology, genetics, structural biology, and pharmacology, this proposal offers an interdisciplinary dissection of the purine salvage pathway of Cryptosporidium parvum, a protozoan parasite that is an opportunistic pathogen of people with AIDS and an NIAID Biodefense Research Category B Priority Pathogen. C. parvum is the etiologic agent of cryptosporidiosis, a debilitating and potentially fatal disease for which no consistently effective chemotherapies exist, especially for immunocompromised individuals. Thus, more efficacious drugs, particularly those that target unique features in C. parvum, are urgently needed. Among the more striking metabolic discrepancies between C. parvum and its human host are the pathways for purine acquisition. C. parvum, unlike mammals, cannot synthesize purines de novo and is dependent upon a unique purine salvage mechanism to fulfill its nutritional needs. Inhibition of purine salvage, therefore, presents a selective therapeutic paradigm for treating C. parvum infections. In this proposal, we will investigate three imperative components of purine salvage; adenosine kinase (CpAK), adenylate deaminase (CpAMPDA), and guanylate synthetase (CpGMPS). There are two Specific Aims. Multi-component Specific Aim I delineates a detailed biochemical, biological, and functional characterization of all three enzymes and a structural analysis of CpAK. We will clone all three genes and functionally evaluate their gene products through biochemical studies of their encoded products and by rescue or complementation of genetic lesions in other organisms. The expression patterns of the CpAK, CpAMPDA, and CpGMPS genes and the levels and location of the three proteins in intact C. parvum parasites will be ascertained throughout biologically accessible life cycle stages. Finally, structure-function studies based upon a molecular model of CpAK will test the roles of key residues on CpAK that are projected to be involved in the catalytic mechanism, and crystallographic studies on CpAK will be initiated with the ultimate intention of determining its three-dimensional structure. The second Specific Aim has two parts. First, we will perform a structure-activity relationship analysis of CpAK against a battery of available nucleoside analogs. Second, we will optimize and implement a high throughput screen of a structurally diverse chemical repository using an E. coli-based assay with the purpose of discovering novel inhibitors of CpAK that could be potentially used for treating the parasitic disease. The "hits" from the screen will be further evaluated using target-based and cell-based assays and eventually in a mouse model that mimics the human disease. Overall, the proposed investigations constitute a logical step in the validation of these enzymes as potential therapeutic targets and in the implementation of a rational strategy of drug discovery for treatment of cryptosporidiosis. PUBLIC HEALTH RELEVANCE The overall purpose of this proposal is to investigate components of the purine salvage pathway of Cryptosporidium parvum, a waterborne parasite that causes a devastating opportunistic infection in people with AIDS and is also a Category B priority pathogen of the National Institute of Allergy and Infectious Disease because the organism can be maliciously manipulated as a biological weapon. There is currently no vaccine and no consistently effective chemotherapy, so there is an urgent need for new drugs and new drug targets. Because purine salvage is essential for Cryptosporidium parvum and not for humans, our studies address the vital issue of validating new targets and discovering potentially novel drugs.

描述（由申请人提供）：合并分子生物学，生物化学，细胞生物学，遗传学，结构生物学和药理学的工具，该提案提供了对隐孢子虫parvum的嘌呤分解途径的跨学科解剖，一种原生动物的寄生虫是一种机会的病原体。患有艾滋病的人和NIAID生物化研究类别B优先病原体。 C. parvum是隐孢子虫病的病因学药物，这是一种令人衰弱和潜在的致命疾病，不存在始终有效的化学疗法，特别是对于免疫功能低下的个体。因此，迫切需要更有效的药物，尤其是那些针对C. parvum中独特特征的药物。在C. parvum及其人类宿主之间的新陈代谢差异中，是嘌呤获得的途径。 C. parvum与哺乳动物不同，无法合成从头开始的嘌呤，并且取决于独特的嘌呤救助机制来满足其营养需求。因此，抑制嘌呤挽救，提出了用于治疗孢子念珠菌感染的选择性治疗范式。在该提案中，我们将研究嘌呤救助的三个命令式组成部分。腺苷激酶（CPAK），腺苷酸化脱氨酶（CPAMPDA）和鸟苷酸合成酶（CPGMP）。有两个具体的目标。多组分特定目的我描述了所有三种酶的详细生化，生物学和功能表征以及CPAK的结构分析。我们将克隆这三个基因，并通过对其编码产物的生化研究以及其他生物体中遗传病变的营救或补充来评估其基因产物。 CPAK，CPAMPDA和CPGMPS基因的表达模式以及完整的C. Parvum寄生虫中三种蛋白质的水平和位置将在整个生物学上可访问的生命周期阶段中确定。最后，基于CPAK分子模型的结构功能研究将测试CPAK上关键残基在催化机制中的作用，并且对CPAK的晶体学研究将启动，以确定其三型三体的最终意图尺寸结构。第二个特定目标有两个部分。首先，我们将对CPAK对一组可用的核苷类似物进行结构活性关系分析。其次，我们将使用基于大肠杆菌的测定法对结构上多样的化学存储库进行优化和实施高吞吐量屏幕，目的是发现可用于治疗寄生虫病的新型CPAK抑制剂。屏幕上的“命中”将使用基于目标和基于细胞的测定法进一步评估，并最终在模仿人类疾病的小鼠模型中。总体而言，拟议的研究构成了验证这些酶作为潜在治疗靶点的逻辑步骤，并且是在治疗隐孢子虫病治疗的药物发现合理策略方面的实施。公共卫生相关性这项提案的总体目的是调查嘌呤孢子虫的嘌呤挽救途径的组成部分，这是一种寄生于艾滋病患者的毁灭性机会感染的水寄生寄生虫，也是B类优先的Allergy和Alerergy和Allergy和Alerergy和Alsergy病原体传染病是因为可以将生物体恶化作为生物武器操纵。目前没有疫苗，也没有一贯有效的化学疗法，因此迫切需要新药和新药靶标。由于嘌呤挽救对隐孢子虫植物而不是人类至关重要，因此我们的研究涉及验证新靶标和发现潜在的新药物的至关重要问题。